The inhibition of FGF receptor 1 activity mediates sorafenib antiproliferative effects in human malignant pleural mesothelioma tumor-initiating cells

Our plain-English summary of The inhibition of FGF receptor 1 activity mediates sorafenib antiproliferative effects in human malignant pleural mesothelioma tumor-initiating cells, by A. Pattarozzi, E. Carra, R.E. Favoni, et al (Stem Cell Research & Therapy, 2017).

The study

• Authors: A. Pattarozzi, E. Carra, R.E. Favoni, R. Würth, D. Marubbi, R.A. Filiberti, L. Mutti, T. Florio, F. Barbieri, A. Daga
• Journal: Stem Cell Research & Therapy
• Published: 2017;8(1):119
• Cited by: 23 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Background: Malignant pleural mesothelioma is an aggressive cancer, characterized by rapid progression and high mortality. Persistence of tumor-initiating cells (TICs, or cancer stem cells) after cytotoxic drug treatment is responsible for tumor relapse, and represents one of the main reasons for the poor prognosis of mesothelioma. In fact, identification of the molecules affecting TIC viability is still a significant challenge.

Methods: TIC-enriched cultures were obtained from 10 human malignant pleural mesotheliomas and cultured in vitro. Three fully characterized tumorigenic cultures, named MM1, MM3, and MM4, were selected and used to assess antiproliferative effects of the multi-kinase inhibitor sorafenib. Cell viability was investigated by MTT assay, and cell cycle analysis as well as induction of apoptosis were determined by flow cytometry. Western blotting was performed to reveal the modulation of protein expression and the phosphorylation status of pathways associated with sorafenib treatment.

Results: We analyzed the molecular mechanisms of the antiproliferative effects of sorafenib in mesothelioma TIC cultures. Sorafenib inhibited cell cycle progression in all cultures, but only in MM3 and MM4 cells was this effect associated with Mcl-1-dependent apoptosis. To investigate the mechanisms of sorafenib-mediated antiproliferative activity, TICs were treated with epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF) causing, in MM3 and MM4 cells, MEK, ERK1/2, Akt, and STAT3 phosphorylation. These effects were abolished by sorafenib only in bFGF-treated cells, while a modest inhibition occurred after EGF stimulation, suggesting that sorafenib effects are mainly due to FGF receptor (FGFR) inhibition. Indeed, FGFR1 phosphorylation was inhibited by sorafenib. Moreover, in MM1 cells, which release high levels of bFGF and showed autocrine activation of FGFR1 and constitutive phosphorylation/activation of MEK-ERK1/2, sorafenib induced a more effective antiproliferative response, confirming that the main target of the drug is the inhibition of FGFR1 activity.

Conclusions: These results suggest that, in malignant pleural mesothelioma TICs, bFGF signaling is the main target of the antiproliferative response of sorafenib, acting directly on the FGFR1 activation. Patients with constitutive FGFR1 activation via an autocrine loop may be more sensitive to sorafenib treatment and the analysis of this possibility warrants further clinical investigation.

In plain English

In this laboratory study, the authors tested sorafenib on cells from human malignant pleural mesothelioma. Of ten cultures from surgical tumor samples in Genova, Italy, four formed tumors in mice. Three (MM1, MM3, MM4, all from epithelioid tumors) were used. Asbestos exposure was not studied. Sorafenib reduced viability in all three. At 48 hours, the concentration halving viability (IC50) was 0.26 micromolar in MM1 and 6.40 and 5.97 micromolar in MM3 and MM4. The drug held more cells in G1 phase in all three. At each culture's IC50 and twice that, it significantly increased apoptosis (programmed cell death) in MM3 and MM4, not MM1; 5 micromolar killed almost all MM1 cells by necrosis. It blocked bFGF-triggered signaling more completely than EGF-triggered signaling, and reduced FGFR1 (bFGF receptor) activation in MM3 and MM4. MM1 cells released more bFGF (304.8 pg/ml) than MM3 and MM4 (5.7 and 9.3 pg/ml). The authors concluded that in these cells sorafenib acts mainly through FGFR1 rather than Raf, its usual main target in other tumors. They noted the small number of cultures and high drug concentrations needed.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory (in vitro) study of patient-derived mesothelioma tumor-initiating cell cultures, kept tumor-forming by growing them in mice
• Who was studied: Ten cell cultures (MM1 to MM10) from post-surgical malignant pleural mesothelioma specimens at IRCCS-AOU San Martino-IST, Genova, Italy. Four formed tumors in mice, and three (MM1, MM3, MM4), all derived from epithelioid tumors, were studied.
• Exposure: The multi-kinase inhibitor drug sorafenib, at increasing concentrations up to 40 micromolar for 24 to 72 hours. The FGFR1 inhibitor PD173074 and the Raf inhibitor AZ628 were used for comparison. No asbestos or fiber exposure was studied.
• Main results:
  • Sorafenib's IC50 at 48 hours was 0.26 micromolar in MM1 cells, and 6.40 and 5.97 micromolar in MM3 and MM4 cells.
  • Sorafenib at IC50 for 48 hours increased the percentage of cells in G1 phase in all three cultures: MM1 from 58.42% to 64.85% (p = 0.037), MM3 from 73.71 to 88.34%, and MM4 from 56.77 to 69.05%.
  • At concentrations equal to each culture's IC50 and twice the IC50, sorafenib increased apoptotic cells compared with untreated controls. In MM3 the increase was from 8.98% to 17.71% and 44.34%. In MM4 it was from 9.41% to 13.6% and 35.57%.
  • In MM1 cells, at its IC50 and twice the IC50, the increase in apoptosis caused by sorafenib was not statistically significant.
  • When the authors tried a higher sorafenib concentration of 5 micromolar in MM1 cells, it was so toxic that almost all the cells died by necrosis, so apoptosis could not be evaluated. The authors did not show these data.
  • The authors linked apoptosis to reduced levels of the survival protein Mcl-1.
  • In all three cultures, sorafenib blocked growth signaling started by bFGF more effectively than signaling started by EGF.
  • In MM3 and MM4 cells, sorafenib reduced bFGF-induced FGFR1 activation by about 75% after 60 minutes.
  • Over 24 hours, MM1 cells released 304.8 pg/ml of bFGF, compared with 5.7 pg/ml for MM3 cells and 9.3 pg/ml for MM4 cells.
  • MM1 cells were the most sensitive to the FGFR1 inhibitor PD173074 (IC50 6.12 nanomolar).
  • In these mesothelioma tumor-initiating cells, the authors judged that direct inhibition of Raf played a minor role in sorafenib's effect. They noted that this contrasts with other tumor types, in which sorafenib is commonly observed to act mainly through Raf.
• The authors' conclusion: The authors concluded that, in these mesothelioma tumor-initiating cells, sorafenib inhibits proliferation and survival mainly by acting on FGFR1, and that the level of FGFR1 activation correlates with drug efficacy. They suggested that patients whose tumors activate FGFR1 through their own bFGF may respond better, a possibility they said warrants clinical investigation.
• Limitations the authors state:
  • Only a small number of patient-derived cultures were analyzed, because surgical samples were hard to obtain and only four of 10 cultures kept their ability to form tumors.
  • The high sorafenib concentrations needed to affect the cells in the laboratory are a problem for applying the results to patients, because kinase inhibitors can have dose-dependent toxic effects.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Lung cancer, Mesothelioma.

Medical terms in this study, explained in our Lexicon: Sorafenib (Nexavar), Pleural Mesothelioma, STEM, Prognosis, Mesothelioma, Apoptosis, Fibroblast.

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• Differential regulation of the sphere formation and maintenance of cancer-initiating cells of malignant mesothelioma via CD44 and ALK4 signaling pathways — Y. Ohno, S. Shingyoku, et al. (2018)
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